Establish an allele-specific real-time PCR for Leishmania species identification

Background Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites. Methods A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay. Results This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients. Conclusions Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40249-022-00992-y.

Traditional Leishmania diagnostic techniques, such as microscopic examination, protozoan culture in vitro and serological immunoassay, cannot identify Leishmania species. In present clinical practice, it is still based on empirical judgment according to the information of local epidemiology. However, it could make inappropriate determination for traveler and co-infections with different species [1]. There are some techniques were developed to discriminate Leishmania species, such as sequencing of individual gene, restriction fragment length polymorphism (RFLP), high resolution melting, multilocus sequencing typing and mass spectrometry [21,[23][24][25][26][27][28][29][30]. As the World Health Organization recommended, the "gold standard" method used to identify Leishmania species is multi-site enzyme electrophoresis (MLEE), which requires culture of parasites [2]. However, some Leishmania species are difficult to culture in vitro with cumbersome experiment procedure which also makes the results among different laboratories incomparable. Although some probe based real-time PCR assays were developed for Leishmania species identification, they are mainly focused on L. mexicana, L. braziliensis, L. peruviania and L. major for CL and not suitable for other common clinical infection related species [31,32]. Thus, for clinical applications, a tool for Leishmania species identification among common clinical pathogens, such as L. donovani, L. infantum, L. major and L. tropica, is needed to be developed using clinical samples directly without isolation and cultivation of parasites.
In this study, to identify Leishmania species, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) genes were selected from 34 housekeeping genes. Our results showed that, HGPRT gene with species-specific single nucleotide polymorphisms (SNPs) can identify parasite species between L. donovani and L. infantum for VL, and SPDSYN gene with species-specific SNPs can distinguish parasite species among L. major, L. tropica and L. donovani/L. infantum for CL. Thus, an allele-specific real-time PCR technique was established for Leishmania species identification with clinical specimens from VL and CL patients.

Patients and samples
A total of 42 clinical samples from patients at Beijing Friendship Hospital, Capital Medical University from July 2015 to Sep 2021 ( Table 1). The bone marrow (n = 22) and skin lesion tissue (n = 20) were collected from patients with VL and CL individually for allele-specific real-time PCR testing. VL patients presented with symptoms such as fever, splenomegaly and/or hepatomegaly, Leishmania amastigotes found in their bone marrow samples under microscope or PCR positive or Leishmania parasite culture positive. CL patients appeared as ulcer and nodule/ plaques features in which Leishmania amastigotes were identified under microscopy. All bone marrow and skin lesion tissue were stored at liquid nitrogen till use. DNA samples of Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi were used as non-leishmaniasis controls.

Potential target fragment selection
Out of 34 genes of Leishmania with sequence polymorphism previously published, 21 were further analyzed according to the inclusion criteria as follows: first, these gene fragments were shown as markers for the molecular characterization of Leishmania strains and species; second, they are common genetic polymorphism sites for the four species (L. donovani, L. infantum, L. major and  Table S1). Each gene sequence among different species of Leishmania parasites were analyzed using MLSTest software (v1.0.1.23, institute de Patologia experimental Universidad Nacional de Salta Argentina, Boston, MA, USA), individually, and genes with sequence polymorphisms and species-specific SNPs were screened out (Additional file 2: Table S2). Furthermore, these sites with speciesspecific SNPs that can be completely distinguished Leishmania species which were selected, specifically, the optimal site that can identify species between L. donovani and L. infantum for VL and distinguishing species among L. major, L. tropica and L. donovani/L. infantum for CL were selected as targets ( Figs. 1 and 2).

Primers and probes design and plasmids construction
Twenty-nine HGPRT sequences from nine different species of Leishmania parasites and 43 SPDSYN sequences from 18 different Leishmania parasites were collected from NCBI database and aligned using BIOEDIT software (v7.0.1, Ibis Biosciences, Carlsbad, CA, USA). Primers were designed based on the conserved region of sequence and probes were designed based on regions with species-specific SNPs of HGPRT genes between L. donovani and L. infantum and species-specific SNPs of SPDSYN genes among L.  Table 2). The sequences of the designed primers and probes were tested against the NCBI nucleotide database using the BLASTn (Basic Local Alignment Search Tool) to confirm the species specificity.

Positive control plasmid construction
The HGPRT fragment of L. donovani and L. infantum, and SPDSYN fragment of L. major and L. donovani/ infantum were amplified from identified clinical specimens and the fragment purified with DNA purification kit (TIANGEN, DP214, Beijing, China). The amplified HGPRT and SPDSYN fragments were ligated into plasmid pUC19 (TAKARA, 3219, Tokyo, Japan) using EcoRI and Hind III sites, individually. The correct cloning of the desired target DNA in the recombinant plasmid was confirmed by PCR amplification and DNA sequencing. Due to lack of L. tropica parasite and clinical samples from patients with L. tropica infection, SPDSYN fragment of L. tropica was synthesized based on sequence (Accession no. KM086079) and ligated into plasmid pUC19 by Sangon Biotech Co., Ltd, and then confirmed by PCR amplification and DNA sequencing.

Analytical sensitivity and specificity of the allele specific real-time PCR for identification of Leishmania species
The limit of detection (LOD) of the allele-specific realtime PCR assay was defined as the minimum number of parasites that could be detected based on 8 repeated tests. We used cultured L. infantum promastigotes enumerated under a microscope and diluted with blood obtained from healthy volunteer as 1,000, 100, 50, 25, 12, 6, 3 or 1 parasites/μl. Total DNA was extracted from each dilution. The LOD was defined based on the experimentally derived assay precision (intra-assay SD < 0.5 and inter-assay CV < 5%). The specificity of the allele-specific real-time PCR assay was tested with other DNA samples obtained from P. falciparum, T. gondii, B. melitensis and O. tsutsugamushi.

Evaluation the performances of allele-specific real-time PCR assay for Leishmania species identification with clinical samples
Total 42 clinical specimens were tested (Table 1), including 22 bone marrow from VL patients and 20 skin lesions from CL patients. These samples were tested according to the standard procedure described above. The amplification products of 42 clinical samples were sequenced with pair ends by Sangon Biotech Co., Ltd. The results of the new method were compared with sequencing method and the consistence was evaluated.

Targets selection for identification of Leishmania species
According to the inclusion criteria described in "Material and methods", 21 genes were screened out from 34 genes, which were previously reported to exhibit sequence polymorphism among Leishmania species (Additional file 1: Table S1). Further analysis indicated that the identity of these 21 genes were 88.3-99.8% among different species and total 1,970 polymorphism sites were observed within them (Additional file 2: Table S2). Our further bioinformatics analysis were performed to select appropriate SNPs from these 1,970 polymorphism sites for Leishmania species identification. The alignment of 29 sequences of HGPRT from nine Leishmania species indicated that two SNPs can distinguish between L. donovani and L. infantum (Fig. 1). Moreover, 1-2 SNPs were found by comparison of 43 sequences of SPDSYN from 18 Leishmania species, which can distinguish Leishmania species among L. major, L. tropica and L. donovani/L. infantum well (Fig. 2). Thus, two potential targets for Leishmania species identification, HGPRT and SPDSYN, were screened out for further investigations.

Development of allele specific real-time PCR assay for Leishmania species identification
To verify the potential application of HGPRT and SPDSYN in Leishmania species identification, the primers and probes were designed according to the conserved sequence of HGPRT and SPDSYN and the SNPs screened out above (Table 2). Firstly, PCRs were performed with template from clinical samples or constructed plasmids. As expected, the primers, VL-HGPRT-F2 and VL-HGPRT-R1 for HGPRT and CL-SPD-F and CL-SPD-R for SPDSYN, can amplify a 145 bp fragment from L. donovani, L. infantum and L. major samples, and 202 bp fragment from L. major, L. tropica, L. donovani and L. infantum samples respectively. In addition, these two pair of primers didn't recognize any DNA from samples of P. falciparum, T. gondii, B. melitensis and O. tsutsugamushi (Fig. 3). These results indicated that the targets we selected here were specific for Leishmania species detection, which were potentially appropriate for further allele-specific real-time PCR assay construction.
Then an allele specific real-time PCR assay for Leishmania species identification were established using the primers and probes described above. Our results showed that this assay can detect 3 parasites/reaction for VL by targeting at HGPRT and 12 parasites/reaction for CL with SPDSYN (Additional file 3: Table S3).
The standard curves of this assay were also obtained using serially diluted plasmid DNA. It showed the PCR efficiency with both single-species and multi-species samples reactions were similar and the amplification curve were coincident as well (Fig. 4). The linear were over a 7-log range with a correlation coefficient (R 2 ) of 0.995-0.999 for VL ( Fig. 4A and B) and 6/7-log range with a R 2 of 0.994-0.999 for CL (Fig. 4C, D and G).
Moreover, both intra-CV% and inter-CV% of Ct values for 20 replicates were < 2% (Additional file 4: Table S4). All these results implied that this allele-specific real-time PCR assay exhibited high precision for VL and CL species identification.

Validation the established Leishmania species identification assay
As the allele-specific real-time PCR assay we developed above exhibited high PCR efficiency and precision, total 42 clinical samples were used to validate the performance of this assay (Table 3). For 22 clinical VL samples, the new method detected 3 as L. donovani infections and 19 as L. infantum, which was consistence with the sequencing results. Similarly, 20 skin lesion CL samples were all identified as L. major using by this new method and confirmed by sequencing as well.
A phylogenetic tree was constructed using 29 Leishmania HGPRT sequences (145 bp) from nine Leishmania species and 22 VL clinical samples. The clustering results shows that 3/22 clinical samples (patient ID 10,11,19) were clustered with L. donovani and 19/22 clinical samples were clustered with L. infantum (Fig. 5 and Table 3). Also, phylogenetic analysis with 43 SPDSYN gene sequences (202 bp) from18 Leishmania species and 20 CL clinical samples indicated that 20 clinical samples were all clustered with L. major ( Fig. 6 and Table 3). Both of these two clustering outcomes were consistence with the new methods we developed here, which further confirmed the reliability of this new assay for Leishmania species identification.

Discussion
In this study, HGPRT and SPDSYN genes, which exhibit species-specific SNPs, were selected based on the screening of 21 housekeeping gene sequences from 9 species of VL and 18 species of CL. According to the conserved regions and species-specific SNPs, primers and probes were designed to perform two allele specific real-time PCR assays respectively. Our results showed that this new developed assay could identify the Leishmania species for VL between L. donovani and L. infantum with HGPRT and for CL among L. major, L. tropica and L. donovani/L. infantum with SPDSYN.
Previous studies identified Leishmania species using a SYBR-green based qPCR followed by melting analysis. Several different target were in these assays, including and 12 parasites/reaction for VL and CL, individually and no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania was detected (Additional file 3: Table S3; Figs. 4 and 5). Considering it takes only 2.5 h to identify Leishmania species directly from clinical samples without parasites isolation or culture, these assays are suitable in clinical practice.
A total of 42 clinical samples (22 VL and 20 CL) were used to evaluate the performance of the allele-specific real-time PCR assay, which identified 22 VL clinical samples as L. donovani (n = 3) and L. infantum (n = 19), 20 CL clinical samples as L. major (n = 20). These results were consistent with the following sequencing analysis, which indicated that these new tools can distinguish SNPs among different Leishmania species well (Table 3). Further phylogenetic analysis was performed to validate the results of these allele-specific qPCR assays, which confirmed their reliability for potential clinical applications (Figs. 5 and 6).
HGPRT gene encoded hypoxanthine phosphoribosyl transferase, which is a central enzyme in the purine recycling pathway of all protozoan parasites [40]. Spermidine synthase encoded by SPDSYN gene is a key enzyme in the polyamine biosynthetic pathway of protozoan parasites [41]. These two housekeeping gene sequences exhibit observed interspecies polymorphism, which imply that our assays in this study could be applied to distinguish not only Leishmania species we described here, but also other species not included in this study. Indeed, our phylogenetic analysis implied that the sequence of HGPRT gene could differentiate more Leishmania species than we tested here, including L. major, L. mexicana complex and Leishmania (Viannia) subgenus (Fig. 5). Meanwhile, SPDSYN gene fragment appears to be able to distinguish Leishmania (Viannia) braziliensis, L. mexicana complex and Leishmania (Viannia) subgenus as well (Fig. 6). Further investigations are worthwhile to be performed to extend the potential scope of these identification assays.
Broad variations are noted in efficacies of leishmaniasis treatment depending on the Leishmania species, which identification would be helpful in clinical practice. For example, antimonial and miltefosine are more effective to L. major and L. donovani than L. infantum [16,42,43]. Unlike L. major, L. tropica appears unresponsive to PM-based ointments [44,45]. Amphotericin B is used to treat L. tropica or L. major related CL, but not L. infantum [46][47][48]. The efficacy rates of azoles for L. infantum, L. donovani, L. major and L. tropica were 88%, 80%, 53% and 15%, respectively [49]. Further, Leishmania species-specific administrations were applied for better clinical efficiency. For L. tropica infection, intralesional treatment was more efficient than intramuscular administration with sodium stibogluconate [50]. Intravenous antimonial treatment could produce better cure rates against L. panamensis or L. braziliensis related CL compared with L. Fig. 4 Quantitative correlation between gene copy number and threshold cycle of the allele specific real-time PCR assay. A, B HGPRT plasmid was serially diluted from 10 2 to 10 8 copies/reaction and subjected to allele specific qPCR. Linear regression of Ct vs lg copy number of HGPRT plasmid were generated using single and multi-species templates for L. donovani (A) and L. infantum (B) detection, individually. C SPDSYN plasmid was serially diluted from 10 2 to 10 8 copies/reaction and subjected to allele specific qPCR. Linear regression of Ct vs lg copy number of SPDSYN plasmid were generated using single and multi-species templates for L. major detection. D, E SPDSYN plasmid was serially diluted from 10 3 to 10 9 copies/ reaction and subjected to allele specific qPCR. Linear regression of Ct vs lg copy number of SPDSYN plasmid were generated using single and multi-species templates for L. tropica (D) and L. donovani/infantum (E) detection, respectively. ΔRn = Rn (normalized reporter)-baseline. Ct Cycle threshold Table 3 The allele-specific real-time PCR results with the samples from 42 patients